Protective role of circRNA CCND1 in ulcerative colitis via miR-142-5p/NCOA3 axis

Background Increasing research indicates that circular RNAs (circRNAs) play critical roles in the development of ulcerative colitis (UC). This study aimed to determine the role of circRNA CCND1 in UC bio-progression, which has been shown to be downregulated in UC tissues. Methods Reverse transcription quantitative polymerase chain reaction was used to determine the levels of circRNA CCND1, miR-142-5p, and nuclear receptor coactivator-3 (NCOA3) in UC tissues and in lipopolysaccharide (LPS)-induced Caco-2 cells. Target sites of circRNA CCND1 and miR-142-5p were predicted using StarBase, and TargetScan to forecast potential linkage points of NCOA3 and miR-142-5p, which were confirmed by a double luciferase reporter-gene assay. Cell Counting Kit 8 and flow cytometry assays were performed to assess Caco-2 cell viability and apoptosis. TNF-α, IL-1β, IL-6, and IL-8 were detected using Enzyme-Linked Immunosorbent Assay kits. Results CircRNA CCND1 was downregulated in UC clinical samples and LPS-induced Caco-2 cells. In addition, circRNA CCND1 overexpression suppressed LPS-induced apoptosis and inflammatory responses in Caco-2 cells. Dual-luciferase reporter-gene assays showed that miR-142-5p could be linked to circRNA CCND1. Moreover, miR-142-5p was found to be highly expressed in UC, and its silencing inhibited LPS-stimulated Caco-2 cell apoptosis and inflammatory responses. Importantly, NCOA3 was found downstream of miR-142-5p. Overexpression of miR-142-5p reversed the inhibitory effect of circRNA CCND1-plasmid on LPS-stimulated Caco-2 cells, and the effects of miR-142-5p inhibitor were reversed by si-NCOA3. Conclusion CircRNA CCND1 is involved in UC development by dampening miR-142-5p function, and may represent a novel approach for treating UC patients. Supplementary Information The online version contains supplementary material available at 10.1186/s12876-023-02641-6.


Introduction
As a chronic disabling inflammatory bowel disease (IBD), ulcerative colitis (UC) generally begins in young adulthood and continues throughout life [1]. The typical symptoms of UC include abdominal pain and diarrhea mixed with mucus and blood. Despite treatment efforts, the etiology and pathogenesis of UC remain unclear [2,3]. Although diagnostic and treatment methods for UC have improved, the prognosis remains poor. In recent years, biomarker-based diagnoses and treatments have received extensive attention [4]. Thus, there is an urgent need to clearly determine the mechanisms of pathogenesis and identify effective biomarkers for UC patients.
Circular RNAs (circRNAs) are a class of non-coding RNA molecules that do not have a 5′ end cap and a 3′ terminal poly(A) tail and form a circular structure via covalent bonds. CircRNAs are formed by back splicing via non-canonical splicing [5]. CircRNAs are a type of RNA molecule without translation ability and have been confirmed to control tumor bio-functions, such as chemotherapeutic resistance, epithelial-mesenchymal transition (EMT), and cell proliferation [6][7][8]. A number of reports have indicated that certain circRNAs are related to the pathogenesis of UC; for instance, circRNA 0001021 regulates UC by sponging miR-224-5p [9]. IL-3 is involved in UC through circular RNA circPan3 [10]. In ulcerative colitis, circRNA Atp9b is overexpressed [11]. CircRNA CCND1 is a new circRNA that has been shown to expedite cell metastasis and proliferation in hepatocellular carcinoma tumorigenesis by regulating the miR-497-5p/HMGA2 axis [12]. In addition, circRNA CCND1 has been reported to be involved in laryngeal squamous cell carcinoma [13]. Although the role of cir-cRNA CCND1 in human cancer has been reported, the underlying mechanism of its regulation in UC is unclear.
MicroRNAs (miRNAs) are conserved small RNAs with lengths of approximately 18-20 bp [14]. Studies have found that many miRNAs are involved in the development of many diseases by post-translational regulation [15,16]. Recently, a number of studies regarding miRNAs in UC have reported a critical role of miRNAs during the development of UC, such as miR-182-5p [17] and miR-29c-3p [18]. Additionally, miRNA-142-5p is involved in cervical cancer [19]. However, the function of miR-142-5p and its related mechanisms in UC have not yet been clarified.
NCOA3 belongs to the SRC/p160 family of nuclear receptor coactivators. NCOA3 directly binds to nuclear receptors and stimulates transcriptional activity in a hormone-dependent manner [20]. Previous research has shown that NCOA3 is associated with the biological functions of several types of human diseases, such as hepatocellular carcinoma [21] and chronic kidney disease [22]; however, a correlation between NCOA3 and UC has not been detected.
Therefore, further exploration of the pathogenesis of UC is necessary for the development of novel treatment strategies with important practical significance. In summary, this study aimed to determine whether circRNA CCND1 regulates the UC process via the miR-142-5p/ NCOA3 axis. Our study identified novel biomarkers for UC treatment.

Methods and reagents
All methods were carried out in accordance with relevant guidelines.

Clinical sample collection
Twenty colonic mucosa samples from patients and healthy individuals were obtained from The First People's Hospital of Lianyungang. Study inclusion criteria were as follows: (1) none of the patients received anti-UC therapy before surgery, and (2) final diagnosis was identified by pathological determination. Exclusion criteria: (1) patients who underwent prior therapy. This study was approved by the Ethics Committee of the First People's Hospital of Lianyungang, and each patient provided written informed consent. The tissues were stored at − 80 ° before use.

Cell cultured
The human colorectal adenocarcinoma cell lines Caco-2 and 293T used for the dual-luciferase reporter-gene assay were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in Ham's F-12 kmedium (Gibco, NY) supplemented with 1% penicillin and streptomycin (Thermo Fisher, USA) and 10% fetal bovine serum (Gibco, NY) in an atmosphere of 5% CO 2 at 37 °C.
Caco-2 cells were treated with LPS (L8880, Solarbio) to establish the UC model in vitro. In brief, Caco-2 cells were incubated with 10 ng/mL LPS for 24 h, and then Caco-2 cells were harvested for subsequent experiments.

Dual-luciferase reporter-gene assay
Wild-type (WT) or mutant type (MUT) sequences of circRNA CCND1 containing putative target sites for miR-142-5p and NCOA3 were also synthesized into the pMirTarget vector (cat. no. PS100062; OriGene Technologies, Inc.) using a luciferase activity assay. Subsequently, 293T cells were co-transfected with circRNA CCND1-WT (or NCOA3-WT) or circRNA CCND1-MUT (or NCOA3-MUT) with mimic control and mimic of miR-142-5p using JETprime, according to the manufacturers instructions (Polyplus, France). Relative luciferase activity was measured using a reporter-gene system 24 h after infection (Promega). The results were normalized to those of Renilla luciferase.

RT-qPCR assay
Following the supplier's instructions, total RNA was recovered from cells using TRIzol ® (Aladdin, Shanghai, China), and cDNA was generated after reverse transcription of RNA with the Titan One Tube RT-PCR Kit (Merck, USA). The expression levels of miRNAs were detected using TransScript ® II Multiplex Probe One-Step RT-qPCR SuperMix UDG (Transgene, China), and RT-qPCR was performed using PerfectStart ® Green qPCR SuperMix (Transgene, Nanjing). Relative expression levels were calculated using the 2 −ΔΔCt method.

Enzyme-linked immunosorbent assay (ELISA)
Supernatant from the cells was harvested and used for the detection of expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8). The ELISA kits were obtained from Beyotime Biotechnology (Shanghai, China). All operations were performed according to the supplier's instructions.

Western blot assay
To collect total protein from cells, radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, China) was used. The proteins were loaded in 10% SDS-PAGE gel and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in PBS-Tween 20 (PBST) solution at room temperature for 1 h, the membranes were incubated with primary antibodies (NCOA3, 1:1000, ab133611, abcam, Cambridge, MA, USA; GAPDH, 1:10000, EA015, ELK Biotechnology, Wuhan, China) overnight at 4 ℃. The membranes were subsequently incubated with the secondary antibody at room temperature for 2 h. Finally, to visualize protein bands, an enhanced chemiluminescence (ECL) substrate (Cytiva, USA) was performed according to the manufacturer's protocol. The original blots were presented in the additional file 1. It should be noted that during the western blot assay, we first cut out the corresponding membrane according to the molecular weight of the target protein prior to hybridisation with antibody, and then the corresponding membrane was incubated the primary antibody. Therefore the original blots is not a full length membranes.

Statistical analysis
The mean ± standard deviation (SD) represents the data from triplicate experiments. Statistical comparisons among groups were performed using Student's t-test or one-way ANOVA followed by Tukey's post hoc test. Statistical significance was set at P < 0.05.

CircRNA CCND1 is down-regulated in UC
Owing to the significant function of circRNA CCND1 in different diseases, this study explored the effect of cir-cRNA CCND1 on UC. We performed RT-qPCR analysis of circRNA CCND1 expression levels in UC. The results revealed that circRNA CCND1 was at a low level in UC samples in contrast to normal tissues (Fig. 1A). CircRNA CCND1 expression was down-regulated in 10 ng/mL LPS-treated Caco-2 cells (Fig. 1B). Collectively, circRNA CCND1 is down-regulated in UC, thus it is involved in the UC process.

MiR-142-5p binds circRNA CCND1
To investigate the potential binding sites of circRNA CCND1 and miR-142-5p, bioinformatic prediction tools (StarBase) were used, and we discovered that miR-142-5p possibly contained circRNA CCND1 binding sites ( Fig. 2A). To confirm the link between circRNA CCND1 and miR-142-5p, a dual-luciferase reporter-gene assay was performed using 293T cells. As shown in Fig. 2B, the relative luciferase level of circRNA CCND1 3ʹ-UTR was notably reduced when cells were co-cultured with a mimic of miR-142-5p. When potential binding sites were mutated, the miR-142-5p mimic exhibited no effect. These results confirmed that miRNA-142-5p is sponged by circRNA CCND1.

Overexpression of circRNA CCND1 repressed cell apoptosis and inflammatory responses via miR-142-5p in LPS-treated Caco-2 cells
To investigate whether circRNA CCND1 functions by inhibiting miR-142-5p expression, Caco-2 cells were co-transfected with circRNA CCND1-plasmid and miR-142-5p mimic. RT-qPCR analysis confirmed that circRNA CCND1 was downregulated in LPSinduced cells and miR-142-5p was significantly upregulated, compared with the control group. In the LPS plus circRNA CCND1-plasmid group, the circRNA CCND1-plasmid enhanced its expression level and knocked-down miR-142-5p levels in contrast with the LPS + control-plasmid group, and miR-142-5p expression levels were restored when the miR-142-5p mimic was co-transfected (Fig. 5A, B). CCK-8 results indicated that the viability of LPS-treated Caco-2 cells was lower than that of the control; however, overexpression of circRNA CCND1 rescued the inhibitory effect of LPS treatment, while co-transfection of the miR-142-5p mimic diminished the phenomenon (Fig. 5C). The flow cytometry assay revealed that LPS led to cell apoptosis, the circRNA CCND1-plasmid transfected into LPStreated Caco-2 cells decreased apoptosis, and co-transfection of miR-142-5p mimic increased the percentage of apoptosis (Fig. 5D, E). The concentrations of TNFα, IL-6, IL-8, and IL-1β were all strongly enhanced by LPS and impaired by circRNA CCND1-plasmid transfection, which was reversed by the miR-142-5p mimic ( Fig. 5F-I).

NCOA3 acts downstream of miR-142-5p
We then tested the downstream mRNA of miR-142-5p using Targetscan 7.0, and found that NCOA3 contained potential miR-142-5p binding sites (Fig. 6A). We used the dual-relative luciferase method to address this binding. We demonstrated that overexpression of miR-142-5p downregulated the luciferase level of the NCOA3-WT reporter gene (Fig. 6B).
NCOA3 was downregulated when NCOA3-siRNA was transfected, in contrast to that in the control siRNA group (Fig. 7B). Furthermore, we found that the inhibition of miR-142-5p increased the mRNA and protein levels of NCOA3 in Caco-2 cells, but NCOA3-siRNA cotransfection reversed this outcome (Fig. 7C, D). These results indicated that NCOA3 is a target of miR-142-5p and that the level of NCOA3 is negatively associated with miR-142-5p.

NCOA3 participated in impact of mir-142-5p silencing in LPS-induced Caco-2 cells
To determine whether miR-142-5p functions by inhibiting NCOA3 expression, Caco-2 cells were co-infected with a miR-142-5p inhibitor and NCOA3-siRNA. RT-qPCR and western blot analysis indicated that miR-142-5p was highly expressed in the LPS-treated model and NCOA3 was down-regulated compared with the control group. In the LPS + miR-142-5p inhibitor group, miR-142-5p inhibitor abolished its expression and restored NCOA3 levels in contrast with LPS + inhibitor control, whereas NCOA3 expression was absent when NCOA3-siRNA was co-transfected ( Fig. 8A-C). CCK-8 results showed that the viability of LPS-induced Caco-2 cells was lower than that of the control group; however, inhibition of miR-142-5p restored the viability of LPS-stimulated Caco-2 cells, whereas co-transfection of NCOA3-siRNA reversed this effect (Fig. 8D). FCS revealed that LPS exposure accelerated the cell apoptotic rate, and miR-142-5p inhibitor transfection of LPS-treated Caco-2 cells could reduce apoptosis, whereas co-transfection with NCOA3-siRNA increased the percentage of apoptotic cells (Fig. 8E, F). The concentrations of TNF-α, IL-6, IL-8, and IL-1β were all notably elevated by LPS treatment and diminished by the miR-142-5p inhibitor, which was reversed by NCOA3 downregulation (Fig. 9A-D).

Discussion
UC is a chronic non-specific IBD characterized by chronic inflammation and ulcerative changes to the intestinal mucosa. UC lesions are primarily located in the mucosa, colonic submucosa, and rectum. However, its pathogenesis remains unclear. Some studies suggest that dysfunction of the immune system is an important factor in UC-induced intestinal inflammation and tissue damage, and others suggest that it may be related to environmental, genetic, infectious, and immune factors.
The clinical treatment of UC is usually based on the use of corticosteroids, immunosuppressants, and aminosalicylates [23][24][25].
In this study, we elaborated on the molecular mechanism by which circRNA CCND1 relieved UC induced by LPS-stimulation. This examination confirmed that cir-cRNA CCND1 relieved UC progression via miR-142-5p, indicating that circRNA CCND1 may serve as a novel biomarker for UC.
Accumulating evidence has shown that circRNAs play a critical role in UC. In UC, circRNAs have been described as biomarkers. circRNA-SOD2 is involved in UC [26]. Xu et al. found that circRNA-HECTD1 promotes autophagy and affects UC [27]. In contrast, circRNA-102,610 is upregulated in UC and promotes EMT via miR-130a-3p [28]. However, few studies have investigated the role of the circRNA CCND1 in UC. In our study, we found that circRNA CCND1 was disturbed in UC. These findings increase the recent understanding of the role of circRNA CCND1 in the modulation of UC, which may be helpful in better understanding the pathological mechanisms underpinning UC.
Many researchers have illustrated that circRNAs regulate disease processes via miRNAs [29,30]. Zhou found that many miRNAs are involved in UC [31]. Li Z indicated that miR-146a and miR-196 are associated with UC [32]. Wu et al. showed that miR-223-3p ameliorates UC via pyroptosis [33]. In contrast, miR-21 and miR-155 repress UC [34]. Here, experimental evidence identified miRNA-142-5p as a downstream target of cir-cRNA CCND1, with confirmed binding sites. We found that the level of miRNA-142-5p was clearly upregulated and was controlled by the level of circRNA CCND1 in UC. Moreover, the data of current study indicated that overexpression of circRNA CCND1 repressed cell apoptosis and inflammatory responses in LPS-treated Caco-2 cells through down-regulating the expression of miRNA-142-5p.
Previously, NCOA3 was reported to play a critical role in human diseases such as breast cancer [35], osteoarthritis [36], hepatic injury, and fibrosis [37]. In this study, we confirmed that NCOA3 was a direct target of miR-142-5p, and it was negatively regulated by miR-142-5p in Caco-2 cells. In addition, the findings revealed that miR-142-5p silencing relieved LPS-induced Caco-2 cells injury through targeting NCOA3, suggesting the important role of NCOA3 in UC development.
This study is the first to clarify the expression of cir-cRNA CCND1 in UC, and clarify the possible molecular mechanism of its involvement in the occurrence and development of UC. It provides more theoretical basis for the pathogenesis of UC, and provides potential targets for clinical treatment of UC. However, our study still has some limitations. For example, this study was mainly conducted on UC cell model, and no in vivo experimental study was conducted. In addition, circRNA CCND1 may also play a role in UC by regulating other signal pathways, so it is also necessary to explore the signal pathways regulated by circRNA CCND1 in UC. We will conduct indepth research on these issues in the next step of research.